BackgroundCompared with a conventional wrist puncture for radial artery catheterization, a distal radial artery puncture has the advantage of reducing the incidence of radial artery occlusion (RAO).AimThe present study was designed to evaluate the feasibility of distal trans-radial access compared with conventional trans-radial access for coronary angiography.MethodsA prospective, randomized, single-blinded, comparative study was conducted at a tertiary care center in India between December 2018 and November 2020. A total of 420 patients (aged >18 years) with signs and symptoms suggestive of coronary artery disease (CAD) and with a palpable radial artery in anatomical snuffbox were randomized into two groups. Group A comprised patients accessed at the distal trans-radial site, and Group B consisted of patients accessed at the conventional trans-radial site for coronary angiography. Baseline demographics, clinical history, and risk factors for CAD were documented. Procedural-related parameters and complications were compared between the two groups.ResultsThe procedural success rate was non-significant between Group A and Group B . Puncture in a single attempt was higher in Group B compared to Group A . There was no significant difference between Group A and Group B for operation time (p=0.207), fluoroscopy time (p=0.183), and contrast volume (p=0.345). The rate of RAO was higher in Group B compared to Group A . Radial artery hematoma/swelling at the puncture site between Group A (10%) and Group B (8%) was not significant (p=0.61). Post-procedural hemostasis time in Group A was 28 ± 7.86 minutes, and in Group B was 24 ± 6.23 minutes. Both post-procedural persistence of pain (p<0.001) and hand clumsiness (p<0.001) were significantly higher in Group B compared to Group A.ConclusionFor coronary angiography, the distal trans-radial access site is a reliable and secure alternate access site. Conventional trans-radial access is the standard method for coronary interventions, diagnosis, and treatment . CompareStudy design and populationThis was a prospective, randomized, single-blinded, comparative study conducted at a tertiary care center in India between December 2018 and November 2020. The study was approved by the Institutional Ethics Committee of LPS Institute of Cardiology (Ec/bmhr/2018/21) on May 21, 2018, and written informed consent was obtained from all the patients before enrollment. A total of 420 patients were enrolled and randomly assigned into two groups: Group A comprised 210 patients in whom distal trans-radial access was used at anatomical snuffbox for coronary angiography, and Group B included 210 patients in whom conventional trans-radial access was used for coronary angiography. Inclusion criteria were (i) Patients aged >18 years; (ii) Patients with signs and symptoms suggestive of coronary artery disease (CAD); (iii) Patients willing to undergo coronary angiography; and (iv) Patients having palpable radial artery in anatomical snuffbox. Exclusion criteria were (i) Patients who had contraindications to contrast agents; (ii) Patients having forearm atrioventricular fistula for hemodialysis; (iii) Patients with a history of coronary artery bypass grafting in whom the radial artery was used as a graft; and (iv) Patients who had type III and type IV radial artery.Data collection and methodologyBaseline demographics, clinical history, and risk factors for CAD were recorded for all the patients. Anthropometric measurements, chest X-ray, electrocardiogram, and 2D echocardiogram were recorded for each patient. Blood and serum investigations were carried out, including complete blood count, blood urea, serum creatinine, coagulation profile, and serum electrolytes.Coronary angiographic procedure-related parameters, including success rate, puncture in a single attempt, puncture time, operation time, fluoroscopy time, and contrast volume, were compared between distal trans-radial access (Group A) and conventional trans-radial access (Group B). The success rate was defined as the number of patients having successful visualization of coronary anatomy. Puncture time was defined as the time from holding a 21-gauge puncture needle in hand to successful radial artery cannulation, confirmed by arterial blood backflow from the radial sheath sidearm. Operation time was defined as time from prepping of skin to compression dressing. Incidences of procedure-related complications such as RAO, RAS, radial artery hematoma/swelling at the puncture site, post-procedural hemostasis time, the persistence of pain, and post-procedural hand clumsiness were recorded and compared between both groups.Distal trans-radial artery puncture techniqueThe patient was positioned with the arm in a neutral position. A normal saline plastic bottle wrapped with a sterile cloth was put under the wrist's ventral aspect, and the patient was asked to hold a medium-sized ball of rolled gauge pieces. This served to keep the distal area open for access by separating the thumb and first finger, which ultimately increased patient comfort. The puncture of the distal trans-radial artery at the distal aspect of the palm was done with the snuffbox region facing upwards rather than the palmar aspect of the wrist. Once the routine disinfection of the skin was done, access was obtained. The distal radial artery was graded into four types based on findings of palpation as follows:Type I Palpable artery without any effort with forceful thrust on the tip of the palmer aspect of the index finger, which is not suppressible with minimal force.Type II Palpable artery without any effort, with forceful thrust on the tip of the palmer aspect of the index finger, which is suppressible with minimal force.Type IIIPalpable artery with effort with weak thrust on the palmer aspect of the index finger.Type IVNot palpable.Our study enrolled patients with type I and type II arteries. After subcutaneous injection of 1.5 mL xylocaine, the artery was punctured, preferably with a 21-gauge open needle, under an angle of 30°-45° from lateral to medial. The needle was directed to the point of the strongest pulse. Accessing the artery at the dorsum of the hand, distal to the tendon of the extensor pollicis longus muscle, was preferred where the artery is usually more superficial than at the snuffbox. A through-and-through puncture was avoided since the needle would touch the periosteum of the scaphoid or trapezium bones, which can be painful. The distal trans-radial artery puncture technique is represented in Figure Conventional trans-radial artery puncture techniqueThe right or left hand was set in the anatomical position, with the arm supinated and the wrist dorsiflexed. A roll gauze or towel was placed under the wrist for a comfortable extension. Subsequently, the access site was disinfected, and 1-2 ml of lignocaine was injected subcutaneously for local anesthesia. Then, the forearm radial artery was palpated to find the point of the strongest pulse. At an angle of 45°, the artery was punctured with a 21-gauge needle, and a 0.021" soft, flexible, metallic wire  was then inserted through the needle. After pulling out the sheath, a compression device  was used for hemostasis. Patent hemostasis protocol was followed by placing a transparent radial artery band at the distal forearm with the placement of a small green box indicator present on the band proximal to the puncture site. Subsequently, 15-19 mL of air was used for the inflation of the band. The patency of the radial artery was checked by palpation and by the color of the palm every 20 minutes. The conventional trans-radial artery puncture technique is illustrated in Figure Statistical analysisData analysis was conducted using the SPSS software version 20 . Quantitative data were expressed as mean ± SD, while categorical data were represented as the number of patients (n) and percentage. The unpaired "t" test was used to compare the quantitative data of the two groups. For qualitative variables, the Chi-square test was employed. A p-value of <0.05 was considered statistically significant.A total of 420 patients were enrolled in this study and randomized into two groups. Group A comprised 210 patients who underwent coronary angiography using distal trans-radial access at the anatomical snuffbox. Meanwhile, Group B consisted of 210 patients where conventional trans-radial access was utilized for coronary angiography. The mean age of patients in Group A was 55 ± 6.8 years, and in Group B was 54 ± 7.2 years (p=0.144). Males were predominant over females among Group A  and Group B . Risk factors, including hypertension (p=0.488), diabetes (p=0.517), hyperlipidemia (p=0.694), and smoking (p=0.769) were non-significant between Group A and Group B. Comparison of baseline demographics and risk factors of CAD among the study population are outlined in Table Procedural success rate was 96% in Group A and in Group B was 98% (p=0.38). The patients in Group A had a significantly lower number of radial artery punctures in the first attempt than those in Group B . Puncture time in Group A was 1.6 ± 0.8 minutes, and in Group B was 1.2 ± 0.6 minutes (p=0.005). Operation time in Group A was 21.0 ± 2.9 minutes, and in Group B was 20.0 ± 3.3 minutes (p=0.207). Fluoroscopy time was higher in Group A than in Group B . The mean contrast volume used was less in Group B (19 ± 5 mL) compared to Group A (21 ± 4 mL). A comparison of procedure-related parameters among the study population is demonstrated in Table Procedure-related complication such as RAO was observed in four (2%) patients in Group A and 27 13%) patients in Group B (p <0.001). RAS was noted among three (1.5%) patients in Group A and among two (1%) patients in Group B (p=1.0). Post-procedural hemostasis time was found to be statistically significant between Group A and Group B . Post-procedural persistence of pain (p<0.001) and post-procedural hand clumsiness (p<0.001) were found to be statistically significant among both groups. A comparison of procedure-related complications among the study population is represented in Table % patientIn the past decade, distal trans-radial artery access at the anatomical snuffbox has emerged as a novel alternative to conventional trans-radial artery access for coronary angiography and interventions. In the present study, we compared the feasibility and safety of this novel distal trans-radial access with conventional trans-radial access for coronary angiography. In our study, the procedural success rate of distal trans-radial access (96%) was similar to that of conventional trans-radial access (98%). Echoing our findings, Sharma AK et al., in their study, also reported no superiority between distal and conventional radial access . HoweverMany studies have shown that distal trans-radial access results in a decreased rate of RAO ,10-13. TIn our study, higher post-procedural hemostasis time was noted in the distal trans-radial access compared to conventional trans-radial access , which could be due to the less established occlusive device and unfamiliarity with the technique. On the contrary, in an earlier study by Vefalı V and Sarıçam E, hemostasis time was higher in traditional trans-radial access compared to the distal trans-radial access  .Post-prStudy limitationsThis study has some limitations. First, this was a single-center study. Second, the sample size was small. Third, puncture of the distal trans-radial artery can be challenging as its diameter is smaller than the radial artery. A steeper learning curve is required for its access, particularly when the anatomical snuffbox's pulse is weak.Conventional trans-radial access and distal trans-radial access techniques had comparable procedural success rates, RAS, and radial artery hematoma/swelling at the puncture site. Lower rates of complications such as RAO, post-procedural persistence of pain, and post-procedural hand clumsiness were observed among patients who underwent coronary angiography via distal trans-radial access. However, a puncture in a single attempt was prominent among patients accessed via a conventional trans-radial artery. Thus, distal trans-radial access is a feasible and safe alternative for coronary angiography and interventions. However, multicentric studies with larger sample sizes, including heterogeneous populations, are warranted to validate and generalize the findings of our study.


Morquio A disease is a genetic disorder resulting in N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency, and patients are currently treated with enzyme replacement therapy via weekly intravenous enzyme infusions. A means of sustained enzyme delivery could improve patient quality of life by reducing the administration time, frequency of hospital visits, and treatment cost. In this study, we investigated poly(ethylene-glycol) (PEG) hydrogels as a tunable, hydrolytically degradable drug delivery system for the encapsulation and sustained release of recombinant human GALNS (rhGALNS). We evaluated hydrogel formulations that optimized hydrogel gelation and degradation time while retaining rhGALNS activity and sustaining rhGALNS release. We observed the release of active rhGALNS for up to 28 days in vitro from the optimized formulation. rhGALNS activity was preserved in the hydrogel relative to buffer over the release window, and encapsulation was found to have no impact on the rhGALNS structure when measured by intrinsic fluorescence, circular dichroism, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In vivo, we monitored the retention of fluorescently labeled rhGALNS in C57BL/6 albino mice when administered via subcutaneous injection and observed rhGALNS present for up to 20 days when delivered in a hydrogel versus 7 days in the buffer control. These results indicate that PEG hydrogels are suitable for the encapsulation, preservation, and sustained release of recombinant enzymes and may present an alternative method of delivering enzyme replacement therapies that improve patient quality of life. Mucopolysaccharidosis IVA  is an autosomal recessive disorder characterized by a functional deficiency in the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme . It occuThe treatment of Morquio A disease primarily focuses on the reduction in GAGs buildup and the management of symptoms. The gold-standard treatment for this patient population is a weekly intravenous (i.v.) infusion of the recombinant human GALNS enzyme (rhGALNS), also known as enzyme replacement therapy (ERT) ,8. This There is an urgent need to develop novel drug delivery methods that can address the limitations of the current treatment for Morquio A patients. Improvement areas include the distribution of rhGALNS at a low and sustained dose and new protocols that can decrease the immune response to rhGALNS. Using hydrogel delivery devices, such as poly-ethylene glycol (PEG) hydrogels, presents an interesting approach. PEG polymers are already widely used for in vivo treatments and are FDA-approved for use in medical devices ,13. In tOur previous study demonstrated that active rhGALNS can be encapsulated within PEG hydrogels and can be slowly released over one week and uptaken by deficient cells in vitro . Here, ww/v) for hydrogels made of an eight-arm PEG-Ac macromer and PEG-diSH crosslinker [p = 0.051). A higher polymer concentration led to faster gelation for any pH tested. However, at the highest pH of 7.4, no further decrease in the gelation time was noted between 25% and 30% w/v polymer concentrations. Overall, there was a significant difference between different polymer concentrations (p = 0.007).Here, we tested several hydrogel formulations for their effect on hydrogel gelation and degradation in vitro . Gelatiosslinker   [We next tested hydrogels prepared with the four-arm PEG-Ac and varying ratios of the crosslinkers PEG-diSH  and PEG-diester-dithiol  B. As bef = 0.46) . Howeverp = 0.02). A subset of these experiments was performed by preparing the precursor solution in a syringe and injecting it into the microcentrifuge tube to mimic the shear stress of a subcutaneous injection , all gels had completely degraded after 48 h. Complementing the effect of the number of arms in the macromer, we showed that increasing the polymer concentration increased the longevity of the gels in vivo  and a PEG-diSH crosslinker  or an eight-arm PEG-VS macromer  and a PEG-DD2 crosslinker . By 48 h, the gels made with VS + DD2 were completely absent, while gels made with Ac + diSH were still present, albeit had decreased in size. As expected, the gels formed from non-degradable polymers (VS + diSH) did not show signs of degradation over the time of the experiment. Likewise, gels made with a combination of non-degradable and degradable crosslinkers degraded noticeably slower.All of the hydrogels described above were degradable via ester hydrolysis, and some of them degraded faster in vivo than expected from prior in vitro experiments ,20. To sw/v. For rhGALNS in solution, we noted a quick and steady decrease in enzyme activity over time, as expected. Interestingly, the activity of rhGALNS incubated in buffer that also contained PEG gels was higher than that in buffer that did not contain gels. After an initial burst release, the enzyme activity  reached a plateau at 3 h (fastest release) for the 10% w/v and at 5 h for the 20% w/v gels made with four-arm PEG-Ac (w/v and at 96 h for the 20% w/v (slowest release) for the gels made with eight-arm PEG-Ac  and ending at 96 h post-gelation, we periodically collected release buffer samples and measured the enzyme activity of these releasates . As contm PEG-Ac A. A platm PEG-Ac B. By ~5–sulation C.w/v four-arm PEG-Ac + PEG-diSH (Gel) and also dissolved in a solution containing 15% w/v uncrosslinked PEG polymer (PEG-OH) and exposed to 0–3 M GuSCN denaturant (λmax) from approximately 340 nm (native) to 355 nm (unfolded), with intermediate wavelengths representing an equilibrium between the two states per the two-state unfolding model. The free energy in each denaturant concentration was calculated from the equilibration constant, and the linear portion was extrapolated to zero denaturant concentration to determine the free energy of unfolding  indicates the increase in the surface area of rhGALNS upon unfolding. The ΔG of PEG-containing samples trended higher than that of the buffer control, although the effect was not statistically significant. If crowding with PEG-OH or confinement within the PEG hydrogel had a significant stabilizing effect on rhGALNS, we would expect a more compact unfolded conformation and a lower m-value [m-value between Gel, PEG-OH, and Buffer, suggesting that the gel did not affect the stability of the rhGALNS tertiary structure upon encapsulation.The stability of the tertiary structure of rhGALNS was quantified by the free energy of unfolding (ΔG) measured via intrinsic fluorescence. rhGALNS was encapsulated in 15% naturant A. rhGALNnfolding B. The sl m-value . Howeverw/v in HEPES buffer, 40 mM reactive groups) and PEG-DM , and two thiol crosslinkers, PEG-diSH  and DTT . rhGALNS incubated in HEPES buffer and 10% w/v PEG-OH (6 kDa) served as controls. The samples were incubated for 1 h at room temperature and analyzed by non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)  A. As we DS-PAGE) B. The hiw/v four-arm PEG-Ac + PEG-diSH hydrogels to those of rhGALNS in buffer  spectra of rhGALNS released from 15% n buffer . GALNS ww/v four-arm PEG-Ac + PEG-diSH), we injected Alexa 680-labeled rhGALNS at a concentration of 5 mg/kg of body weight. We found that non-encapsulated rhGALNS was eliminated from the body at 7 days post-injection, while hydrogel-encapsulated rhGALNS was present until 23 days post-injection  with a molecular weight of 66.5 KDa as a model stable protein. Alexa 680-labeled BSA (10 mg/kg of body weight) was encapsulated in four-arm PEG-Ac hydrogels, and its fluorescence was compared to that of control non-encapsulated BSA. We found that there was a significant difference in intensity after 24 h of subcutaneous injection into the left flank of mice. Non-encapsulated BSA was eliminated from the mice within 24 h, while the signal of the BSA encapsulated in the PEG hydrogel was present until 10 days post-injection . Maintainjection A,B. To enjection C,D, highThe overall goal of our work is to develop an injectable PEG hydrogel delivery system that can encapsulate rhGALNS, retain its activity, and release it in a sustained manner over 7–28 days. While we have optimized degradable hydrogels for drug delivery in previous works, this approach has not been studied for the delivery of enzyme replacement therapies ,20. At aWe chose PEG hydrogels due to their excellent biocompatibility and tunability ,13. MichWe also examined the effect of the reaction pH, the PEG concentration, and the choice of crosslinking reaction groups on the degradation time in vivo. The hydrolytic degradation of Michael-type addition thioester bonds both drives protein release from the gel and allows the gel to be cleared from the body. Corroborating our prior in vitro work , we obsem-value, an indication of the change in the surface area between the folded and unfolded states [m-value in the gel, as the hydrogel mesh would restrict conformations with a large surface area and encourage a more compact unfolded state [w/v four-arm PEG-Ac (10 kDa) + PEG-diSH (3.4 kDa), have a mesh size of 8.5 nm, or approximately 2–3 times the hydrodynamic radius of the rhGALNS monomer  to encapsulate fluorescently labeled proteins . When not encapsulated, rhGALNS was cleared in 7 days, which is consistent with a study that suggests that GALNS is retained intracellularly for 7 days [To confirm our release window in vivo, we used the optimal in vitro hydrogel formulations  was obtained from Alfa Aesar . PEG-diacrylate , PEG-dimethacrylate , four-arm polyethylene glycol-acrylate , eight-arm polyethylene glycol-acrylate , four-arm polyethylene glycol-vinylsulfone , eight-arm polyethylene glycol-vinylsulfone , and polyethylene glycol-dithiol  were purchased from Laysan Bio Inc. . The polyethylene glycol diester-dithiol (PEG-DD2) crosslinker was synthesized in-house as previously described ,20. HEPECHO cells were transfected to constitutively express rhGALNS, as described previously . Cells wrhGALNS activity was assayed as described previously ,19. Briew/v. Polymer solutions were mixed in a 1:1 molar ratio between the Michael-Type addition donor groups of the crosslinkers (PEG-diSH or PEG-DD2) and the acceptor groups of the multi-arm PEG macromers . The mixtures were left to initiate gelation within the syringe and injected immediately prior to fully solidifying. PEG polymers were dissolved separately in 0.1 M HEPES buffer at pH values ranging from 6.5 to 7.4, in concentrations ranging from 10% to 30% C57BL/6J mice (4 weeks old) or C57BL/6 albino mice (7 weeks old) were selected for all the experiments in this study. All mice were housed in a pathogen-free environment with a normal diet. All procedures were in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines under approved protocols at Saint Louis University and followed the NIH’s Guide for the Care and Use of Laboratory Animals .Subcutaneous injections of hydrogels were performed on 4-week-old mice. rhGALNS-free hydrogel mixtures were prepared as described above. Prior to fully solidifying, hydrogel mixtures were subcutaneously injected into the left and right flack of each mouse. Mice were sacrificed after 24, 48, 72, and 96 h post-injection, and the remaining gels were removed for a size comparison.rhGALNS was encapsulated into the gels by mixing 4000 units of the rhGALNS enzyme in the hydrogel macromer solution prior to combining with the crosslinker solution, as described above. Gels were formed into uniformly shaped slabs by pipetting 30 µL of the mixture onto a parafilm-coated glass plate and covering the gel with another parafilm-coated plate 1 mm apart (separated by silicon spacers).Following encapsulation, hydrogels were incubated in 270 µL of rhGALNS release buffer  and incubated at 37 °C with shaking. Sampling was performed by removing 30 µL of the release buffer and replacing it with an equal volume of fresh buffer. Immediately after collection, rhGALNS activity was assayed for these samples. Initial collections were performed at the start of the experiment, as well as at 1, 3, and 5 h following incubation. Further sample collection occurred every 24 h, which was extended to every 48 h after 10 days of incubation. To assess the preservation of enzyme activity, hydrogels were prepared for each time point (a separate hydrogel for each time point) as mentioned above, and the entire releasate sample was removed. The releasate samples were immediately placed at −80 °C for storage until testing or used immediately for further analysis of enzyme activity, as described previously.Hydrogel gelation time was measured by the inverted tube method . Brieflyw/v), and PEG-diSH (20% w/v) were prepared in 0.1 M HEPES, pH 7.4. The stock solutions were then used to prepare a hydrogel precursor solution of 50 µg/mL rhGALNS and 15% w/v PEG with stoichiometrically equal acrylate and thiol groups. The hydrogel precursor solution was pipetted into UV-star clear bottom 96-well plates at 70 µL per well. After 60 min of gelation, 95 µL of the denaturant  was added to each well and mixed on a Fisher orbital shaker  at 200 rpm for 2 h. Then, the intrinsic fluorescence intensity  of rhGALNS in the presence of the denaturant was measured on a Spectra Max M5 with SoftMax Pro Software . The energy of unfolding was calculated from solvent denaturation curves using the linear extrapolation method [To measure the free energy of unfolding, hydrogels were prepared as described above with some minor modifications. Briefly, stock solutions of rhGALNS (0.5 mg/mL), four-arm PEG-Ac , PEG-diSH (10 kDa), PEG-DM (1 kDa), and PEG-OH (6 kDa) for up to 24 h at room temperature. After incubation, the samples were prepared according to the manufacturer’s protocols for non-reduced SDS-PAGE. PAGE gels were run at a constant 200 V for 40 min on a Bis-Tris 4–12% gradient gel in an XCell SureLock cell . The lanes were loaded with a sample volume corresponding to 0.4 µg of rhGALNS. The gels were stained with SimplyBlue Safestain.rhGALNS at 40 µg/mL was incubated in HEPES buffer (control), 0.15% The secondary structure of rhGALNS released from PEG hydrogels was measured using a JASCO 715 Circular Dichroism Spectrophotometer . Briefly, rhGALNS was concentrated or diluted with release buffer to 0.1 mg/mL prior to measurements. Far UV spectra (190–260 nm) were obtained with a 1.0 nm bandwidth, 0.5 nm step interval, and 1.5 s/nm scanning speed. The secondary structure was calculated using the BeStSel online CD spectra analyzing tool .w/v four-arm PEG-Ac or eight-arm PEG-Ac + PEG-diSH, loaded with BSA or rhGALNS. Hydrogel mixtures (200 µL) were subcutaneously injected into the left flank of each mouse. The mice were anesthetized with isoflurane, and fluorescent images were acquired on an IVIS Spectrum imager  every 24 h for up to 24 days.First, 7-week-old male C57BL/6 albino mice received a single subcutaneous injection of fluorescently labeled BSA or rhGALNS. The proteins were labeled with ALEXA 680  according to the manufacturer’s instructions. Precursor solutions were prepared to reach a final concentration of 20% 2). Analyses were conducted with a Holm–Šídák’s multiple comparison test. p < 0.05 was considered statistically significant.All IVIS images were acquired under the same field of view using autoexposure. The data were analyzed using Living Image software (version 4.5). Fluorescent regions were quantified and expressed as Radiant Efficiency (p/s/sr)/(µW/cmξ) of the hydrogels was determined according to the Flory–Rehner theory, as described previously [G), after swelling in HEPES buffer at pH 7.4 for 24 h (MS), and after drying in a 60 °C oven overnight (MD). The swelling ratio, Qm, which is the ratio of swollen mass to dry mass (MS/MD), was then used to calculate the mesh size per Equation (1) [s2, is the polymer volume fraction in the swollen state , Cn is the characteristic ratio for PEG, CM is the average molecular weight between two adjacent crosslinks, rM is the molecular weight of a PEG repeat unit, and l is the average bond length between the C-C and C-O bonds in a PEG repeat unit.The mesh size  .(1)ξ= to be feasible for subcutaneous injection. A high PEG concentration and a high number of PEG arms also resulted in the longest degradation times due to their higher crosslink density. However, we observed degradation rates that were faster than our previous in vitro studies and hypothesize that a low crosslink density, irregular gelation, and mechanical stimulation in vivo may be accelerating the breakdown of the gel. We encapsulated rhGALNS in the PEG hydrogels and observed a retention in enzymatic activity relative to the buffer for up to 28 days. However, the presence of blank PEG gels alongside unencapsulated rhGALNS also preserved activity, suggesting that stabilization may result from the interaction between rhGALNS and the PEG backbone rather than encapsulation shielding the protein. Likewise, the encapsulation of rhGALNS did not significantly alter its free energy of unfolding compared to the buffer or solutions of an uncrosslinked PEG polymer. While a PEGylation reaction was observed between PEG-acrylate and rhGALNS, there was no change in the secondary structure of the released rhGALNS, as measured by CD. Lastly, we administered fluorescently labeled rhGALNS to mice via subcutaneous injection and observed the presence of rhGALNS for up to 20 days when delivered in a hydrogel, compared to 7 days for rhGALNS in the buffer solution. Together, these results indicate that PEG hydrogels are a suitable drug delivery system for preserving and releasing active rhGALNS. The tunability of the Michael-type addition gelation reaction allows for a formulation tailored for both rhGALNS activity and a gelation time optimized for subcutaneous injection. The PEG hydrogel preserves and releases active rhGALNS over a time window significantly longer than that of the current standard treatment, although future studies are needed to compare the efficacy of the treatments.A.M.M. and S.P.Z. are inventors on the U.S. Patent No. 11,034,950 related to this work.


Playing football is associated with a high risk of injury. Injury prevention is a priority as injuries not only negatively impact health but also potentially performance. Various multi-component exercise-based injury prevention programs for football players have been examined in studies.We aimed to investigate the efficacy of multi-component exercise-based injury prevention programs among footballers of all age groups in comparison to a control group.We conducted a systematic review and meta-analysis of randomized and cluster-randomized controlled trials. CINAHL, Cochrane, PubMed, Scopus, and Web of Science databases were searched from inception to June 2022. The following inclusion criteria were used for studies to determine their eligibility: they (1) include football (soccer) players; (2) investigate the preventive effect of multi-component exercise-based injury prevention programs in football; (3) contain original data from a randomized or cluster-randomized trial; and (4) investigate football injuries as the outcome. The risk of bias and quality of evidence were assessed using the Cochrane Risk of Bias Tool and the Grading of Recommendations Assessment, Development, and Evaluation (GRADE), respectively. The outcome measures were the risk ratio (RR) between the intervention and the control group for the overall number of injuries and body region-specific, contact, and non-contact injuries sustained during the study period in training and match play.Fifteen randomized and cluster-randomized controlled trials with 22,177 players, 5080 injuries, and 1,587,327 exposure hours fulfilled the inclusion criteria and reported the required outcome measures. The point estimate (RR) for the overall number of injuries was 0.71  with very low-quality evidence. The point estimate (RR) for lower limb injuries was 0.82  with moderate-quality evidence; for hip/groin injuries, the RR was 0.56  with low-quality evidence; for knee injuries, the RR was 0.69  with low-quality evidence; for ankle injuries, the RR was 0.73  with moderate-quality evidence; and for hamstring injuries, the RR was 0.83 (95% CI 0.50–1.37) with low-quality evidence. The point estimate (RR) for contact injuries was 0.70  with moderate-quality evidence, while for non-contact injuries, the RR was 0.78  with low-quality evidence.This systematic review and meta-analysis indicated that the treatment effect associated with the use of multi-component exercise-based injury prevention programs in football is uncertain and inconclusive. In addition, the majority of the results are based on low-quality evidence. Therefore, future high-quality trials are needed to provide more reliable evidence.PROSPERO CRD42020221772.The online version contains supplementary material available at 10.1007/s40279-022-01797-7. The stuIn the present study, we included all controlled, multi-component exercise-based injury prevention programs containing at least two or more exercises. Players of the intervention group performed these programs during their training sessions in addition to their usual training and were compared to a control group. Criteria for study inclusion were: (1) include football (soccer) players; (2) investigate the preventive effect of multi-component exercise-based injury prevention programs in football; (3) contain original data from a randomized or cluster-randomized trial; and (4) investigate football injuries as the outcome. Studies were excluded from the meta-analysis if they were: (1) studies with a single exercise intervention; (2) studies with a primary target on performance or other physical measurements than injuries; (3) studies using protective equipment  as part of the intervention; and (4) studies published in a language other than English.Possible studies were identified using a systematic search process. First, we searched the following databases CINAHL, Cochrane, PubMed, Scopus, and Web of Science from the earliest record to June 2022, with the following search strategy: (injury prevention OR warm-up program OR neuromuscular program OR f-marc OR 11 +) AND . The reference lists of the studies recovered were hand searched to identify potentially eligible studies missed by electronic searches. Two reviewers independently  performed the selection of studies based on the title and abstract provided by the bibliographic databases. The full-text evaluation followed on those selected studies from the first selection step. A third reviewer (RO) was responsible for resolving any discrepancies in the selection process.For each eligible study, four reviewers  extracted data independently using a standardized data extraction form . One secI2 > 50%); (3) indirectness ; (4) imprecision (downgraded by one level if the upper and lower CIs had a > 0.5 difference); and (5) publication bias . Evidence obtained was categorized into four levels of evidence quality: high, moderate, low, and very low [The risk of bias was assessed for each included trial according to the recommendations outlined in the Cochrane Handbook for Systematic Reviews of Interventions . The folvery low  Table 1I2 > 50%;The primary outcome was the risk ratio (RR) for the overall number of injuries. Body region-specific injury RRs for the lower limb, hamstring, hip/groin, knee, and ankle were secondary outcomes. Additionally, the overall number and the region-specific injury RRs were assessed for a non-contact versus contact induced cause. All injuries occurring in official training and match play during the respective study period were included.I2, τ2, and Q value (χ2 test for heterogeneity). We interpreted I2 values according to guidelines by Higgins and Green, a low heterogeneity for I2 values between 25 and 50%, a moderate f heterogeneity or 50–75%, and a high heterogeneity for ≥ 75% [If studies did not report RR estimates, we converted them to RRs as far as possible , 26. Outor ≥ 75% . A smallor ≥ 75% . Statistn = 4986), 2968 studies remained. After screening the titles and abstracts, 69 full-text articles were left. A further 54 studies had to be excluded as they did not present data on injuries, included non-football players, or were neither cluster RCTs nor RCTs. Finally, 15 articles were included in the meta-analysis  studies had a high risk of bias in two or more domains. The domain “other bias” was the most frequent cause for a high risk of bias within the studies (46%), with seven studies neither reporting an intention-to-treat analysis nor an adjustment for clustering .I2 = 80.5%; τ2 = 0.067; p < 0.001). The width of the 95% PI suggests that the effect in future similar studies lies between 0.38 and 1.32  of 0.71  with moderate-level evidence (downgraded one level because of a risk of bias). For knee injuries, the RR was 0.69  with low-level evidence (downgraded one level because of a risk of bias and one level because of inconsistency). For hip/groin injuries, the RR was 0.56  with low-level evidence (downgraded one level because of a risk of bias and one level because of imprecision). For hamstring injuries, the RR was 0.83 (95% CI 0.50–1.37) with low-level evidence (downgraded one level because of a risk of bias and one level because of imprecision). With regard to ankle injuries, the RR was 0.73  with moderate-level evidence (downgraded one level because of a risk of bias). For each calculation, the 95% PI was wider in comparison to the 95% CI.Regarding the secondary outcome analyses, i.e., the body region-specific injury risk Fig. , the poiI2 = 67.3%; τ2 = 0.100; p = 0.016), with evidence rated as low level (downgraded one level because of a risk of bias and one level because of inconsistency). Additionally, the point estimate (RR) for contact injuries was 0.70 , with moderate-level evidence (downgraded one level because of a risk of bias). The width of the 95% PI suggested that the effect may vary from being very protective to an increased risk of injury for both outcomes, i.e., non-contact injuries (95% PI 0.55–1.10) and contact injuries (95% PI 0.40–1.24)  of 0.78 . In female football players, the point estimate (RR) was 0.82  .Regarding a distinction between male and female individuals, the point estimate (RR) for the overall number of injuries in male football players was 0.70 , in youth, the RR was 0.74 , in seniors, the RR was 0.73 , and, in veterans, the RR was 0.91 (95% CI 0.53–1.57) .The point estimate (RR) for the overall number of injuries in children was 0.52 , knee (RR 0.69 vs IRR 0.52), and ankle injuries (RR 0.73 vs IRR 0.68) was lower in our study, but comparable for hip/groin injuries (RR 0.56 vs IRR 0.59). A likely explanation for the differing results between the reviews is that we included a higher number of studies that examined different types of programs in the analysis. An additional explanation could be the inclusion of studies with children because injury patterns vary with age [Riley et al.  suggestewith age . The moswith age . Moreovewith age , we inclwith age , which sFor the first time, this study investigated the effect of multi-component exercise-based injury prevention programs not only on non-contact injuries but also on contact-related injuries. The point estimate (RR) for contact injuries was 0.70 (95% CI 0.56–0.88). Surprisingly, the estimated risk reduction was higher than for non-contact injuries for which the vast majority of programs are designed. Most programs include strength exercises that mostly focus on core stability. Furthermore, plyometrics  are often part of the programs. They have the potential to improve lower leg strength, functional leg stability, and balance, thus improving the ability to absorb external forces, for example, induced by contact. The 11 + Kids  program The subgroup analysis showed a point estimate (RR) of 0.70 in male football players. These results mimic the data of the Al Attar et al. study . HoweverRegarding female individuals, the pooled results showed a point estimate (RR) of 0.82. This result falls within the range of results reported by studies with similar inclusion criteria , 16. HowThe subgroup analysis for age groups showed a point estimate (RR) of 0.52 in children, a RR of 0.74 in youth, 0.73 in seniors, and 0.91 in veteran football players. The point estimate in youth and seniors is homogeneous with the current available evidence , 41. TheI2 = 80.5%, which should be interpreted as high heterogeneity according to the Cochrane Handbook for Systematic Reviews of Interventions [In the current analysis, we calculated the PIs for the main investigated outcomes. Prediction intervals were wider in comparison to confidence intervals. Based on this evidence, there is a lack of compelling data to affirm the certainty of preventive effects from multi-component exercise-based injury prevention programs. However, for our meta-analysis, we have to take into account that the use of PIs has its shortcomings. IntHout et al.  mentioneventions . In addiventions  stated tTo the best of our knowledge, this review is the first to analyze the efficacy of multi-component exercise-based injury prevention programs among footballers of all age groups. One strength of this systematic review is that it included multiple analyses. It investigated the risk reduction for the overall number of injuries as well as of body region-specific, contact, and non-contact injuries. Subgroup analyses for age and sex were also performed. Additionally, the PIs for the main outcomes were calculated. A further strength is the large number of participants , injuries (5080), and exposure hours  included in comparison with other reviews –16. FurtI2 = 80.5%). The lack of information about compliance with the prevention program in many studies is another limitation of this review. Furthermore, there was missing information on content and compliance with the usual warm-ups/training routines of the control groups. Another limitation is the high risk of bias, especially from the “other bias” domain, with seven studies failing to report the use of an intention-to-treat analysis and of an adjustment for clustering. Finally, two deviations  from the original study protocol have to be mentioned as limitations of this review.However, this review also has some limitations, mainly that > 50% of the reported effects were based on studies with a very low or low level of evidence. The main outcome variable provided high heterogeneity among the studies , which appears crucial to reduce injury risk. Currently, a large number of different exercises are included because it is unknown which exercises (or which combination of them) are most effective in general or in relation to specific injuries. Tailoring the exercises would potentially mean fewer injuries and more efficiency.This meta-analysis indicated that evidence for the meaningful effects of multi-component exercise-based injury prevention programs in football remains inconclusive at best. This statement is based on PIs that were wider than the frequently employed CIs, with a range from very protective effects to an increased injury risk. In addition, the quality of evidence is a major issue in existing studies. These findings call for future high-quality trials to provide more reliable evidence regarding the efficacy of injury prevention programs in football.Supplementary file1 (PDF 226 KB)Below is the link to the electronic supplementary material.


This study aimed to compare the antero-lateral and posterior localized renal masses in laparoscopic partial nephrectomy with the retroperitoneal approach in terms of operative, functional, and oncological outcomes. Patients who underwent retroperitoneal laparoscopic partial nephrectomy by a single surgeon between January 2013 and January 2021 were included in the study. A one-to-one propensity score matching (PSM) analysis was conducted to obtain two balanced groups. The patients were divided into two groups as posterior and antero-lateral according to the localization of the mass. A total of 239 patients were included in the PSM analysis, with 65 patients allocated to each group. The mean operative time was 79.2 ± 11.2 min in the posterior group, while it was 90.0 ± 11.6 min in the antero-lateral group (P < 0.001). Warm ischemia time was 15.9 ± 2.4 min in the posterior group and 18.6 ± 2.7 min in the antero-lateral group (P < 0.001). The median decrease in eGFR at 1 year was 4.8  mL/min in the posterior group and 5.0  mL/min in the antero-lateral group (P = 0.219). The warm ischemia time and clamping technique were found to be significant factors for predicting eGFR change after surgery . We report that retroperitoneal laparoscopic partial nephrectomy provided longer warm -ischemia and operative time for antero-lateral renal masses than posterior masses. However, long-term oncological and functional results were similar for both localizations. Partial nephrectomy (PN) is strongly recommended as a standard approach in patients with cT1 renal tumors as it has better functional outcomes and similar oncologic outcomes when technically suitable compared to radical nephrectomy (RN) . Also, iDepending on the surgeon’s experience and tumor location, either transperitoneal (TP) or retroperitoneal (RP) approach is preferred –5. The bTherefore, in our study, we aimed to compare the perioperative, functional, and oncological results for patients with posterior and antero-lateral renal masses treated with LPN by a single surgeon experienced in the RP approach.Following institutional ethical committee approval, clinical data collected for 259 consecutive patients with localized renal masses who underwent retroperitoneal LPN by a single surgeon (M.A.) in two centers between January 2013 and January 2021 were retrospectively evaluated. Patients with congenital renal malformation (n = 1), solitary kidney (n = 2), coagulation disorders (n = 9), and radiofrequency ablation history (n = 1), and those with missing data (n = 7) were excluded. A total of 239 patients were included in the analysis.The demographic and clinical characteristics of patients including data such as age, gender, body mass index (BMI), American Society of Anesthesiologists (ASA) score, comorbidities (such as diabetes and hypertension), smoking status, estimated glomerular filtration rate (eGFR), and tumor size were recorded. Preoperative creatinine levels were measured routinely 3–10 days before the surgery.Preoperatively, abdominal CT scan and/or MRI were taken for all patients in order to assess tumor characteristics and clinical TNM staging. The R.E.N.A.L.  nephrometry scoring system was used to assess the tumor complexity for each renal mass. The anterior and posterior distinction of tumor localization was made according to the R.E.N.A.L. nephrometry scoring previously described in the literature . AnotherAs with previous descriptions of the RP–LPN technique, under general anesthesia and in the full flank position, the RP cavity was bluntly entered using the index finger with a 16–18 mm transverse incision under the tip of the 12th rib in Petit’s triangle, then the cavity was expanded using an optical dilatation balloon . A 12 mmPerioperative and postoperative data of the patients, including operative time (OT), warm ischemia time (WIT), open surgery conversion rate, estimated blood loss (EBL), pre- and postoperative hematocrit values, length of hospital stay (LOS), final pathology, pathological stage, surgical margin involvement, and complications were recorded and compared according to tumor location. The classification of recorded complications was made according to the Clavien-Dindo system . eGFR waFinally, the validated M.I.C. system  was used to evaluate the success rate of partial nephrectomy. In addition to this trifecta system, the condition of the patient was evaluated 1 year later using the pentafecta system, which is defined as >90% preservation of eGFR and no upgrading of chronic kidney disease until 12 months after LPN.Data are expressed as n (%), mean ± standard deviation, or median and interquartile range , as appropriate. The normality assumption was checked with the Kolmogorov–Smirnov test. The Mann–Whitney U test or Student’s t-test was used for continuous variables. Pearson chi-square or Fisher’s exact test was performed for categorical variables. Multivariate linear regression analysis was performed to identify predictive factors for GFR changes after surgery. Potential imbalances between groups were expected because of the study design. A one-to-one propensity score matching (PSM) analysis was conducted to reduce potential bias and to attain comparable groups. A caliper of 0.20 of the standard deviation of the logit of the propensity score was used to obtain similar groups regarding the set of covariates. Statistical assessments were performed using SPSS software pack  and R program (version 2.15.2 for Windows). To incorporate these programs and to perform PSM analysis, a developer-based software providing a custom dialog in the SPSS menu was used . P < 0.0A total of 130 patients, 65 with posterior and 65 with antero-lateral renal masses, were included in the analysis after PSM . The disThe mean operative time was 79.2 ± 11.2 min in the posterior group and 90 ± 11.6 min in the antero-lateral group (P < 0.001). Warm ischemia was applied to 41 (63.1%) patients in both groups. The mean duration of warm ischemia was 15.9 ± 2.4 min in the posterior group, while it was 18.6 ± 2.7 min in the antero-lateral group (P < 0.001). In three procedures, which started with off-clamping in the antero-lateral group, warm ischemia was reversed due to intraoperative bleeding. None of the patients in either group converted to open surgery or radical nephrectomy. Clavien-Dindo Grade 1 complications were observed in five patients of the posterior group and three patients of the anterolateral group. For the anterior group, four out of five patients had transient elevation of serum creatinine and one patient had fever. For the antero-lateral group, three patients had transient elevation of serum creatinine. Due to the decrease in hemoglobin in the postoperative period, blood transfusion (Clavien-Dindo Grade 2) was required in one patient in the posterior group and two patients in the antero-lateral group. A double J stent was inserted (Clavien-Dindo Grade 3a) after urine extravasation from the collecting system in one patient in the antero-lateral group. The results for intraoperative, perioperative, postoperative, and early complications are presented in When the final pathology results were evaluated, surgical margins were positive in four (4.3%) patients in the posterior group and four (5.6%) patients in the antero-lateral group (P = 0.706). The histological types were clear cell carcinoma for 92 (70.8%), papillary carcinoma for 19 (14.6%), oncocytoma for 8 (6.2%), chromophobe for 5 (3.8%), angiomyolipoma for 3 (2.3%), and cystic-solid for 3 cases (2.3%). Recurrence was detected in one patient in both groups during mean follow-up of 51.1 ± 15.8 months.The median absolute change in eGFR at 1 year was 4.8  mL/min in the posterior group and 5.0  mL/min in the antero-lateral group (P = 0.219). Warm ischemia time and clamping technique were found to be significant factors for predicting eGFR change after surgery  .The retroperitoneal approach for laparoscopic partial nephrectomy is chosen less often by urologists, in spite of reporting lower complications, hospital stay, and operative duration . Though Transperitoneal access is widely preferred in patients with anterior renal masses because it allows a large working area , 4. HoweIn a study comparing retroperitoneal robot-assisted partial nephrectomy (RP-RAPN) for posterior and -antero-lateral masses, the RP approach was identified to be a safe and effective method for posterior masses in addition to -antero-lateral masses . LimitatIn the study by Harris et al., they compared the transperitoneal approach in RAPN for antero-lateral and posterior masses . They reAnother retrospective two-center comparison of TP-RAPN and RP-RAPN reported that surgical durations were statistically significantly in favor of RP-RAPN . PentefaVariation in GFRs after partial nephrectomy is a basic parameter for assessment of functional outcomes. The targeted functional outcome is eGFR change of less than 10% after partial nephrectomy . FactorsOur study also had some limitations. The main limitation was its retrospective design, which may have led to the possibility of misclassification bias. The relatively small sample size was also a major limitation. However, in order to compensate for potential cofounders and preoperative imbalances, we performed PSM analysis. Additionally, the follow-up duration was also sufficiently long.RP-LPN is known to provide many advantages for posterior renal masses. In this study, we report that although the operative time and warm ischemia time are longer for antero-lateral masses compared to posterior-located masses, RP-LPN is also an effective and safe approach for antero--lateral masses in terms of oncological and functional outcomes.HA contributed to the project development, data collection, data analysis, and manuscript writing and editing. AG was involved in project development and manuscript writing and editing. AY contributed to the project development and data collection. SA helped with data collection and manuscript writing and editing. KK was involved in data collection and manuscript writing and editing, and MA contributed to the project development and manuscript writing and editing.


Erwinia amylovora (E. amylovora), a highly infectious bacterial pathogen responsible for fire blight, which is notorious for its rapid spread and destructive impact on apple and pear orchards. This method allows for ultra-sensitive measurements without pre-amplification or labeling compared to conventional methods. To detect a single cell of E. amylovora, we used Lipopolysaccharide Transporter E (LptE), which is involved in the assembly of lipopolysaccharide (LPS) at the surface of the outer membrane of E. amylovora, as a capture agent. We confirmed that LptE interacts with E. amylovora via LPS through in-house ELISA analysis, then used it to construct the sensor chip by immobilizing the capture molecule on the sensor surface modified with 3′-Aminopropyl triethoxysilane (APTES) and glutaraldehyde (GA). The LptE-based SIS sensor exhibited the sensitive and specific detection of the target bacterial cell in real time. The dose–response curve shows a linearity (R2 > 0.992) with wide dynamic ranges from 1 to 107 cells/mL for the target bacterial pathogen. The sensor showed the value change (dΨ) of approximately 0.008° for growing overlayer thickness induced from a single-cell E. amylovora, while no change in the control bacterial cell (Bacillus subtilis) was observed, or negligible change, if any. Furthermore, the bacterial sensor demonstrated a potential for the continuous detection of E. amylovora through simple surface regeneration, enabling its reusability. Taken together, our system has the potential to be applied in fields where early symptoms are not observed and where single-cell or ultra-sensitive detection is required, such as plant bacterial pathogen detection, foodborne pathogen monitoring and analysis, and pathogenic microbial diagnosis.Herein, we developed a bio-functionalized solution-immersed silicon (SIS) sensor at the single-cell level to identify  E. amylovora is a Gram-negative, rod-shaped bacterium belonging to the family Enterobacteriaceae [E. amylovora bacterium, has rapidly disseminated across various apple- and pear-producing regions, which is now present in more than 50 countries, including North America, New Zealand, and Europe [E. amylovora exhibits remarkable resilience to environmental stressors such as freezing, drying, and heat [E. amylovora, a plant disease, has led to its categorization as a quarantine organism in numerous countries. The movement of plants and their derivatives from areas impacted by fire blight are strictly monitored by plant quarantine regulations. [eriaceae ,2. Sinced Europe . The bacd Europe ,5. Notaband heat ,7. The glations. .E. amylovora [E. amylovora in standardized laboratories, but the time required to obtain results is prolonged, and the sensitivity to certain pathogens is constrained, with some pathogens being uncultivable [E. amylovora. Real-time PCR is a powerful tool for the diagnosis of plant diseases, but it has the disadvantage of requiring sample preparation for nucleic acid extraction and additional probe synthesis [E. amylovora cells has been demonstrated as a promising approach [E. amylovora [E. amylovora detection, we confirmed that our sensor system possesses novelty in recognition element, rapid response time and ultra-high sensitivity. A comparison of various diagnostic methods for E. amylovora detection is described in Studies have attempted to develop biosensors for the detection of mylovora . Traditiltivable . Real-tiynthesis ,12. The approach . Nonethemylovora ,15. Howemylovora ,17. Nonep-polarized wave of the probing beam, that is the Brewster angle, the ellipsometric signal (Ψ) shows ultra-sensitive to overlayer thickness variations while the phase difference (Δ) almost remains unchanged. These factors reduce the noise signals induced from surrounding conditions, which allows for high signal-to-noise ratio on the affinity analysis of target antigens. In the current study, a bio-functionalized SIS sensor has been fabricated by immobilizing the LptE protein of E. amylovora as a capture agent on the silicon surface, enabling extremely sensitive detections with real-time monitoring manner for E. amylovora bacteria at the single-cell level.A promising sensor platform based on silicon, called SIS sensor, has been developed that can replace the traditional gold film used in Surface Plasmon Resonance (SPR) sensors ,21. The 3′-Aminopropyl triethoxysilane (APTES) and glutaraldehyde (GA) were obtained from Sigma Aldrich . Silicon wafer (100) as the SIS sensor chip was purchased from MCL Electronics Materials , and 11.5 mm × 11.5 mm sensor chips were used for this work. Phosphate-buffered saline  served as the binding and washing buffer.E. amylovora strain used in this study was obtained from KRIBB. E. amylovora was cultured by inoculating a single colony onto LB agar   and subsequently transferring it to LB medium. The bacterial suspension was adjusted to an OD600 of 0.1 by dilution with LB medium and then incubated at 28 °C under shaking (220 rpm) conditions. Changes in absorbance at 600 nm were monitored until the bacteria entered the stationary phase after 24 h.The E. coli  was used as host, and pET-21a (+)  was used as vector for the expression of LptE. Restriction enzymes and DNA-modifying enzymes were purchased from Promega  and New England Biolabs . In order to clone the full-length gene encoding for LptE in E. amylovora, it was amplified with the forward primer (ACCATATGCGACATCCGATAGTCTCT) and the reverse primer (ACCTCGAGTCGGGTGTAGGAGTTGTC) via PCR. The resultant DNA fragment was then inserted into the pET-21a (+) plasmid using the NdeI/XhoI restriction enzyme cleavage sites, and the plasmid DNA was transformed into BL21 (DE3) cells for expression.BL21 (DE3) E. coli cells were grown in a 10 mL LB starter culture overnight at 37 °C. One mL of the starter culture was inoculated in 100 mL of LB with ampicillin. Cells were grown at 37 °C with shaking until OD600 = 0.6. Cells were induced with 1 mM isopropyl-2-D-thiogalactopyranoside (IPTG)  and grown for 4 h. Cells were then harvested by centrifugation at 6000× g at 4 °C for 10 min. Harvested cells were resuspended in 50 mM Tris-HCl buffer (pH 8.0), and disrupted by sonication. The crude cell lysates were separated into total, soluble, and insoluble fractions, which were analyzed by 12% SDS-PAGE. In order to purify the recombinant proteins, 10 mL of the crude cell lysates were loaded onto an IDA-miniexcellose affinity column . The recombinant proteins were subsequently eluted with 5 mL of 0.5 M imidazole in the same buffer . The protein concentration was determined by Nano drop . Finally, recombinant LptE protein was concentrated to 100 mg/mL, and stored at −80 °C for further experiments.g for 10 min at 4 °C. The resulting LPS samples were purified to remove any remaining particulates and stored in sterile vials at 4 °C.LPS was extracted using an LPS extraction kit  with some modifications. Briefly, cells were harvested and lysed in lysis buffer (50 mg of cells/mL of lysis buffer) and then subjected to a vigorous vortex to dissolve cell clumps. After the addition of chloroform, the sample was centrifuged for 45 min at 4 °C. The upper aqueous layer was collected, and 2 volumes of purification buffer were added to 1 volume of aqueous layer. The mixture was then incubated at −20 °C for 2 h, before centrifugation at 20,000× 4 cells/well of E. amylovora in PBS for 20 min at room temperature. Serial dilutions from LptE proteins in PBS, covering a wide range of concentrations from 0.1 to 15 ug/mL, were prepared and added. Following an hour of incubation at 37 °C, three washes with PBS were performed followed by incubation with primary antibody (His-tag antibody) for 1 h at 37 °C. Then, three washes with PBS were performed followed by the addition of HRP-conjugated secondary antibody (anti-mouse IgG). After the final three washes with PBS, the plate was developed with 100 uL/well of peroxidase substrate for 5 min, and 50 uL/well of H2SO4 was added as a stop solution. The optical density was measured at 450 nm in an ELISA plate reader.Microplates were coated overnight at 4 °C with 75 uL/well of 1 ug/mL poly-L-lysine  diluted in 0.05 M bicarbonate ELISA coating buffer. The poly-L-lysine-modified microplate was incubated with 10In order to immobilize LptE onto a silicon surface, a series of surface modifications were performed. Firstly, the silicon chips were washed using ethanol solution to remove organic contaminants and particles, and then the sensor surface was modified with a 2 vol% APTES in ethanol for 12 h. The self-assembled monolayer of APTES was treated with a 2 vol% solution of GA in PBS for 2 h. Following this, the APTES-GA-modified surface was utilized for bio-functionalization with LptE as a capturing agent.2 surfaces, and scanned with a multimode Nanoscope V controller. AFM images were obtained in the tapping mode under ambient conditions using the Igor Pro 6.36 program. The height and the roughness were determined from horizontal line scans .For the AFM analysis, LptE  was dried on top of the APTES-GA-modified SiOThe LptE-modified SIS sensor system used a p-type silicon wafer (100) cut to 11.5 mm × 11.5 mm as a sensor chip applied to the sensor cell tilted 2° to the prism surface. The PBS was allowed to flow for about 2–5 min until the sensor signal stabilized. The incident angle of the probing light was automatically adjusted at the Brewster angle with respect to the sensor chip using the goniometer  mount on the SIS system which can provide a high accuracy of 0.001°. One-hundred ug/mL of LptE protein  was prepared and utilized in this study, as optimized in our previous study, and the flow rate was 30 uL/min. The sensor layer was washed thoroughly with PBS solution to remove unbound or weakly bound LptE proteins on the surface. The target samples were injected over the bio-functionalized SIS surface, and unbound bacterial pathogens were removed using PBS for 5 min. The reactive values for target bacteria were subtracted by the non-specific signals and were analyzed by averaging the measured signals for 3 min at the washing step.Ψ, Δ); Ψ and Δ are the amplitude ratio and phase difference between p-polarized and s-polarized lights as electric fields aligned in parallel and perpendicular, respectively, to the plane of incidence. These parameters are defined by the complex reflectance ratio ρ asp-polarized and s-polarized light; Ψ = tan−1 (|ρ|); and Δ = δp − δs. The SIS sensor measures at a non-reflecting condition for the p-polarized wave where Ψ shows sensitivity for the thickness change while Δ is almost constant; therefore, Ψ is used as a sensing parameter which detects the variation of surface thickness due to the binding events on the sensor surface.The SIS sensor is a polarizer–sample–analyzer (PSA) system that measures the characteristics of a target analyte by analyzing changes in the polarization state of light that is reflected from a sample with a specific polarization state. The SIS sensor operates sensitively to ellipsometric parameters  for 24 h. The properties of bacterial growth were recorded under shaking culture conditions (180 rpm). As shown in E. amylovora showed two phases, the log or exponential phase (0–20 h) and stationary phase (20–24 h), which is consistent with a previously reported bacterial population growth curve [E. amylovora was calculated, as the number of E. amylovora was defined as follows: OD600 0.1 = 1 × 108 cells/mL. The E. amylovora yielded a cell count of approximately 7 × 108 cells/mL at 20 h after incubation, and remained at that level.th curve . Based oE. amylovora, as a capture agent for the first time. The significant benefit of using LptE as a capture agent lies in its capability to recognize bacteria directly from samples without the need for prior amplification. In addition, to enhance the detection performance, it is possible to utilize modifications of LptE. For example, LptE can be engineered through directed molecular evolution screening to increase its affinity for its cognate LPS. Nevertheless, the detection of E. amylovora using LptE requires comprehensive studies, including the optimization of the protocol and the assessment of different strains and plant samples, to evaluate the practicality and reliability of this diagnostic method.The map of recombinant pET-21a (+)-LptE expression vector is presented in E. amylovora, an in-house ELISA analysis was performed. For this purpose, a whole-bacterial-cell ELISA using bacterial cells as antigens was developed. In particular, poly-L-lysine was introduced onto the surface of microplate wells to immobilize bacterial cells, based on the notion that the cationic polypeptide interacts with the negatively charged cell surface via ionic adsorption. As shown in E. amylovora was calculated as Kd = 1.44 ug/mL (R2 = 0.960), showing the affinity between LptE and the target bacterial cells. LptE can interact with bacterial cells via LPS recognition. LPS-based ELISA analysis showed that changes in signal intensity were proportional in response to the concentration of the tested LptE ranging from 0.1 to 10 ug/mL (data not shown), indicating the association of LptE with LPS, the major component of the outer membrane of Gram-negative bacteria. The results imply that LPS-binding protein can be useful for the diagnosis and monitoring of Gram-negative bacteria.To validate the interaction between LptE and E. amylovora bacterial cell detection, LptE was applied to the sensing area of SIS sensor for 3 min, followed by a subsequent 1 min PBS buffer wash to eliminate unbound molecules. As shown in 2 surface served as the baseline, resulting in a marginal alteration of Ψ signal due to the thickness variation of the chip, approximately 0.0221. Upon immobilization of LptE onto the APTES-GA-modified silica surface, the Ψ value exhibited a substantial increase to approximately 0.2307, and the binding level remained consistent during the washing step. The experimentally obtained Ψ value of 0.2307 can be converted to an estimated surface thickness of approximately 1.0185 nm. Given that LptE protein forms a roll-like structure, this value aligns with the estimated molecular size of LptE .The immobilization of LptE as the capture molecule was achieved on silicon surfaces modified with APTES-GA. For the implementation of the sensor chip to 2, APTES-GA and APTES-GA-LptE deposited on the SIS silica surface. Software for AFM  was used to calculate the average surface roughness  of each AFM image. Rq values were determined as follows: 0.21 nm for bare SiO2, 0.61 nm for APTES-GA-SiO2, and 2.69 nm for APTES-GA-LptE-SiO2, demonstrating the binding of the protein of interest on the SIS surface.The SIS surface modified with LptE was imaged through AFM to investigate the roughness of the LptE layer. 2). In this configuration, the buffer solution serves as the incident medium, whose role is to provide a constant temperature and humidity environment; errors due to refractive index changes are extremely eliminated at the Brewster angle, resulting in a sensitivity more than 10 times higher than conventional ellipsometric sensors [E. amylovora and the LptE-functionalized SIS surface. 2 > 0.992) for wide dynamic range between 100 and 107 cells/mL. The coefficient of variation for triplicate measurements was within 21.0% at the bacterial detection point.The most important feature of ellipsometry as a sensing technique of the SIS sensor is the simultaneous measurement of refractive index and thickness, which can provide accurate detection, even for very low-molecular-weight or low-concentration biomolecules on the sensor surface. Specifically, the SIS sensor operates by directing incident light through a buffer solution, a bio-layer, and a substrate layer  using the following conversion equation:Ψ by 0.2265 within the optimized conditions. According to the data shown in Ψ = 0.008° for a 35.32 pm thickness change. If we consider the average size of E. amylovora to be around 5 µm in diameter, the volume of the bacterial cell can be calculated to be approximately 65 femtoliters. Assuming that the volume corresponding to a single cell is passivated on the SIS sensor cell (1.5 mm × 6.0 mm) and interacted with the inclined beam with a major axis of <6.54 mm and a minor axis of <2.0 mm, the thickness is calculated to be approximately 7.22 pm. If target substances are evenly distributed on the surface, an overall change in refractive index across the surface is measured. However, in the case of single-cell adhesion to the surface, where a single and large analyte is distributed at a specific point, the change in refractive index at that point will be much greater. Therefore, it can be speculated that the reason why the experimental data differ from the calculated value is that the refractive index change can vary depending on the adsorption state and location of the analytes. In order to evaluate the specificity of the LptE-SIS sensor, we performed SIS measurement in response to B. subtilis, a Gram-positive bacterium that does not contain LPS. As shown in Ψ value of B. subtilis showed a negligible increase of approximately Ψ = 0.0003, which is only about 3.7% of the increment observed in E. amylovora. Although specificity analysis for various bacterial strains was not performed in the current study, this result demonstrates that at least the ellipsometric angle of the LptE-modified SIS sensor is not affected by LPS-free B. subtilis.At the Brewster angle, in our case at 72.18°, the change in S sensor , the dΨ E. amylovora , non-covalently linked with LptE, is unable to endure such conditions and readily dissociates from the surface. As shown in 7 cells/mL E. amylovora, washed at a low flow rate (30 uL/min) and regenerated at a high flow rate (100 uL/min), was repeated five times over 10,000 s for continuous signal measurement of SIS in response to E. amylovora. The observed magnitude of SIS signal change in the first reusability test was 0.0149, and consistent signal peaks were maintained without signal reduction until the fifth reusability test. From the results obtained from the reusability tests, it was confirmed that the sensor could be reused for the detection of E. amylovora through surface regeneration modified with LptE, which is advantageous in terms of the cost-effectiveness aspect of the sensor.Another important aspect of biosensors is the sensor’s surface regeneration. Biosensors are often designed for disposable measurements, and to reuse a biosensor for bacterial detection, a complex surface/interface architecture needs to be employed followed by regeneration. Regeneration typically involves the removal of bacterial cells and underlying linker layers and capture agents on the sensor surface, along with surface modifications to facilitate the re-binding of bacteria. This process is often complex and time-consuming. Therefore, we attempted to investigate the surface regeneration and reusability potential of the SIS sensor modified with LptE utilizing a physical regeneration method involving an enhanced flow rate, which can avoid hindrance to the binding capacity of receptor proteins due to the use of a regeneration buffer containing glycine . This apE. amylovora based on different isothermal amplification methods [4 cells/mL for LAMP, 103 cells/mL for LAMP-CRISPR/Cas, and 102 cells/mL for recombinase polymerase amplification (RPA) and RPA-CRISPR/Cas. To our knowledge, the single-cell detection of E. amylovora has not been reported before. It is assumed that the excellent outcome obtained from this work is due to the integration of an ultra-sensitive SIS sensor and a bio-receptor capable of recognizing LPS from bacteria. In general, bio-functionalized sensors exhibit higher sensitivity with smaller sizes of bio-receptors. This is because smaller bio-receptors can detect subtle changes at the molecular level and respond to smaller energy changes. This study employed a relatively large protein, LptE, with a molecular weight of around 25 kDa, as a capture agent for bacteria. If domain function analysis can identify the minimal size necessary for LPS binding within the LPS-binding domain (LBD), the resulting bio-receptor could potentially exhibit a faster binding rate and higher sensitivity, thereby serving as an ideal capture agent. Therefore, the next study will focus on bacterial detection using a pre-functionalized SIS sensor with a minimal LBD.Recently, Ivanov et al. reported a comparison of the detection limit for  methods . In thatE. amylovora. In this study, an ultra-sensitive bio-receptor-based SIS sensor capable of detecting E. amylovora at the single-cell level has been developed. LptE, a lipopolysaccharide-binding protein capable of recognizing the glycolipid on the bacterial surface, was used as the capture agent to capture E. amylovora. The bacteria-detecting LptE was found to interact with E. amylovora through in-house ELISA testing, and this was confirmed to be the result of direct binding between LptE and its cognate LPS. The bio-functionalized SIS sensor exhibited a wide dynamic range with linearity (R2 > 0.992) spanning from 100 cells/mL to 107 cells/mL, highlighting its capability to detect E. amylovora at the single-cell level, with no observed signal change for the positive Gram bacterium (B. subtilis). Furthermore, the LptE-modified SIS sensor demonstrated effective reusability for the continuous detection of E. amylovora. The great advantage of using the LptE-modified SIS is that genetically engineered receptors with improved affinity and specificity for the target material can be easily produced. Therefore, a superior bio-functionalized SIS can be introduced by designing a more effective bio-capture agent for the target bacteria. In field tests where immediate bacterial detection without the need for culturing pathogenic bacteria is imperative, there exists a critical demand for highly sensitive sensors. In this context, the current study, which has achieved the single-cell detection limit for E. amylovora, sheds light on the potential applicability of our system in the field of primary field screening requiring high-sensitivity detection and large-scale sample monitoring.To prevent the spread of fire blight and minimize its damage, there has been an increasing demand for early detection of the causal agent,


T-2 toxin is a mycotoxin routinely found as a contaminant of cereal grains worldwide. A portable mass spectrometer was adapted to enable the detection of T-2 toxin in wheat and maize by APCI-MS. In order to facilitate rapid testing, a rapid cleanup was used. The method was able to detect T-2 toxin in soft white wheat, hard red wheat, and yellow dent maize and could be used to screen for T-2 at levels above 0.2 mg/kg. The HT-2 toxin was only detectable at very high levels (>0.9 mg/kg). Based on these results, the sensitivity was not sufficient to allow the application of the screening method to these commodities at levels recommended by the European Commission. With a cut-off level of 0.107 mg/kg, the method correctly classified nine of ten reference samples of wheat and maize. The results suggest that portable MS detection of T-2 toxin is feasible. However, additional research will be needed to develop an application sensitive enough to meet regulatory requirements. Fusarium sambucinum species complex, that commonly infest crops +. As a result, the background signal for HT-2 toxin and the associated standard deviation in this signal in the matrix was much greater than for T-2 toxin. This itself was enough to negatively impact the LOD and LOQ for HT-2, which, of course, are based on these statistics. Attempts to tune the instrument to better detect HT-2 came at the expense of the detection of T-2 (data not shown). The tune parameters selected were done to optimize the detection of both. Nevertheless, the detection of HT-2 was poor.The poorer response of the portable MS to HT-2 was significant, as the European Commission regulates both toxins in grains . The cau13C-T-2 as an internal standard  provides assessments used by policymakers in setting acceptable levels within the European Union (EU). An assessment of T-2 and HT-2 toxins established a tolerable daily intake of 0.02 µg/kg body weight per day and an acute reference dose of 0.3 µg . In thisThe practical application of the portable TD-APCI-MS at levels that meet the full range of EU regulatory requirements requires further improvements in sensitivity. Perhaps this will come with improvements to the linear ion trap upon which the instrument is based. Currently, that appears to be a significant technical challenge. As an alternative, the required sensitivity may be attained by using an instrument capable of MS/MS. Transportable MS/MS instruments are available but are significantly bigger and heavier (less portable) than the instrument used herein. Reducing the size of MS/MS instruments may ultimately solve the issues that were encountered here. An alternative solution may be to use a small, portable chromatography system to introduce samples. The resulting decrease in matrix interferences would likely contribute to improved sensitivity. Reduction of the matrix could also be achieved with a different form of sample preparation: for example, immunoaffinity cleanup, which was used successfully for the measurement of fumonisins in corn . HoweverA portable mass spectrometer was adapted to enable the detection of T-2 and HT-2 toxins in wheat and maize by APCI-MS. In order to facilitate rapid analysis of these toxins, a rapid cleanup was used. The method was able to detect T-2 and HT-2 in soft white wheat, hard red wheat, and yellow dent maize. The method can be used to screen for T-2 at levels that are greater than 0.2 mg/kg. However, sensitivity was not sufficient to allow the application of the method to the screening of these commodities at levels recommended by the European Commission. Attaining the sensitivity to meet such criteria will likely require further advances in instrumentation, such as portable MS/MS, improvements in sensitivity to the linear ion trap used here, or the incorporation of a more extensive cleanup, such as an immunoaffinity column cleanup.13C-T-2 toxin were purchased from Biopure. Reference materials of wheat or maize containing established levels of T-2 and HT-2 toxins were also purchased from Trilogy. These included four samples of wheat flour with non-detectable T-2 and HT-2 (LOD 0.005 mg/kg), one sample of wheat flour contaminated with both toxins, and four samples of ground maize contaminated with both toxins. A single sample of contaminated oat flour was obtained from FAPAS . Details of these samples are listed in Except where noted otherwise, deionized water  was used in the preparation of all reagents. HT-2 toxin in solid form was obtained from Sigma Chemical, dissolved to approximately 1 mg/mL in ethanol, and the concentration was established by comparison to an HT-2 analytical standard supplied by Biopure . In a similar fashion, solid T-2 toxin was dissolved to approximately 2.5 mg/mL in acetonitrile, and the concentration was established by comparison to a T-2 analytical standard supplied by Trilogy Analytical Laboratory . Standard solutions of v/v) by shaking in a wrist-action shaker  for 30 min. The solution was filtered through a Whatman 2V filter . Then 4 mL of extract was applied to a Romer MycoSep 225 cleanup column, and approximately 2.5 mL of cleaned-up extract was collected. Of this, 1 mL of cleaned-up extract was dried under a gentle stream of nitrogen gas for 15 min at 50 °C. The dried extract(s) were reconstituted with 2 mL of a solution of 12.5 mM ammonium formate prepared in H2O/MeOH . In instances where 13C-T-2 was incorporated as an internal standard, the solution also contained 0.1875 µg/mL of the stable isotope. Reconstituted extracts were filtered through a 0.2 µm pore nylon filter, 13 mm diameter (Cytiva). The purified extracts contained the equivalent of 0.125 g of grain per mL of extract.Samples of whole-kernel wheat or whole-kernel maize were ground to the consistency of flour using a coffee grinder. Twenty-five grams was extracted with 100 mL of acetonitrile/water  or in purified extracts of uncontaminated wheat or maize. Calibrants were prepared over the range of 0.03125 to 1.25 µg/mL T-2 and 0.15625 to 6.25 µg/mL HT-2. Experiments on matrix effects were conducted in quadruplicate. Spiking was performed by adding standard T-2 and HT-2 in acetonitrile to ground wheat or maize. Spiked samples were stored overnight at ambient temperature to allow the solvent to evaporate prior to extraction. For validation of screening tests, the criteria of the European Commission [The extent of matrix on ionization was determined by preparing calibrants in ‘solvent’ . The instrument contains a linear ion trap mass analyzer and contains an inlet suitable for atmospheric sampling. It is self-contained in the sense that it does not require an external supply of gas, as air is used. The source that was purchased was a thermal desorption atmospheric pressure chemical ionization (TD-APCI) unit. The original source was equipped with a single route for sample introduction consisting of a metal inoculation loop a. The loThe inlet of the original APCI source was modified to accept a continuous supply of infused liquid. The spray nozzle from the API source of a Thermo Finnigan LCQ DECA mass spectrometer  was used for this purpose b. The spm/z = 484; mass tolerance: 1; spectrum average: 10, with running average enabled. Spectra were collected over the m/z range from 50 to 850. The instrument was tuned using purified T-2 and HT-2. The finalized tune parameters were: AC start: 3.0; AC stop: 28.0; AC frequency: 362.5; AC phase: 82; Rf level: 100; Trap bias: 2.5; Trap entry: 4; and Trap exit: 32. Spectra were collected in both Profile and Centralized modes. Signals from m/z 484 were used to determine T-2, and signals from m/z 442 were used to determine HT-2.Operating parameters for the mass spectrometer were as follows: positive mode; heater: 200 °C; sample pump: 0% ; ISM HV: +2.8KV, ISM HVS Anode: −1200V. Settings for the mass analyzer were as follows: mass center: 13C-T-2 toxin was incorporated as an internal standard at a concentration of 0.1875 µg/mL. In such cases, relative response ratios were obtained by dividing the signals from T-2 or HT-2 by the 13C-T-2 signal. Calibration curves were constructed using either the un-ratioed (“raw”) data or the relative response ratios to 13C-T-2. Data were fit using a first-order linear regression . The limits of detection (LOD) were determined as the concentration of toxin calculated to give a response three standard deviations above that of control (un-spiked) samples. The limit of quantitation (LOQ) was determined as the concentration calculated to give a response 10 standard deviations above that of control samples.In some experiments, such as for determining the extent of matrix effects, For hard red winter wheat, an additional set of experiments were conducted to determine the Cut-Off value. The Cut-Off value represents the signal at which the rate of false negatives is predicted to be five percent. It was determined according to guidelines developed by the European Commission  and was STC). The one-tailed t-statistic  for 20 replicates was 1.729. The SDSTC was the standard deviation in the signal ratios for the 20 spiked samples.The screening target concentration (STC) was the concentration at which the assay would be used to screen samples. In this case, the STCs were 0.2 mg/kg (T-2) and 1.5 mg/kg (HT-2). Twenty samples spiked at this level were used to obtain an average response for each toxin (R


There are little data on remote monitoring (RM) of implantable loop recorders (ILRs) in patients with unexplained syncope and whether it confers enhanced diagnostic power.To evaluate the effect of RM in ILR recipients for unexplained syncope for early detection of clinically relevant arrhythmias by comparison with a historical cohort with no RM.SyncRM is a propensity score (PS)-matched study prospectively including 133 consecutive patients with unexplained syncope and ILR followed up by RM (RM-ON group). A historical cohort of 108 consecutive ILR patients with biannual in-hospital follow-up visits was used as control group (RM-OFF group). The primary endpoint was the time to the clinician's evaluation of clinically relevant arrhythmias .p = 0.005).The primary endpoint of arrhythmia evaluation was reached in 38 patients (28.6%) of the RM-ON group after a median time of 46 days  and in 22 patients (20.4%) of the RM-OFF group after 92 days (25–368). The PS-matched adjusted ratio of rates of arrhythmia evaluation was 2.53  in the RM-ON vs. RM-OFF group (In our PS-matched comparison with a historical cohort, RM of ILR patients with unexplained syncope was associated with a 2.5-fold higher chance of evaluations of clinically relevant arrhythmias as compared with biannual in-office follow-up visits. Implantable loop recorders (ILRs) are indicated in high-risk patients with unexplained syncope in whom comprehensive evaluation has failed to identify the cause of transient loss of consciousness and in those with suspected reflex syncope and frequent or severe syncopal recurrences . In the We conducted the SyncRM study, a prospective, multicenter, observational investigation including consecutive patients who received an ILR for unexplained syncope, between January 2019 and September 2021 at five investigational clinics (NCT04435262). Approvals from the local Ethics Committees were obtained for all participating sites. All these patients were remotely followed up (RM-ON group) with no prespecified schedule of in-office visits. A historical cohort of consecutive patients who had received an ILR for the same indication of unexplained syncope, between 2017 and 2019, was used as a control group (RM-OFF group), after balancing potential confounders with a propensity score (PS) method. The minimum study follow-up was 18 months.Before undergoing ILR implantation for unexplained syncope and study enrollment, eligible patients had to complete the recommended syncope diagnostic workout, including clinical evaluation, electrocardiogram, echocardiogram, carotid sinus massage, and head-up tilt test .All devices were implanted with standard techniques and programmed to detect atrial fibrillation with >12% RR interval variability, bradycardia episodes with <40 bpm heart rate, high ventricular heart rate episodes with >180 bpm, sudden rate drops >40%, and asystole episodes >3 s. All devices in the RM-ON group were remotely monitored with the Home Monitoring (HM) System  capable of daily transmissions of device data and arrhythmia-related diagnostics. Automatic RM alerts with notifications were activated only for the detection of episodes of asystole, bradycardia, sudden rate drop, and ventricular tachycardia. Detailed settings are reported in Patients or the public were not involved in the design, conduct, reporting, or dissemination plans of our research.This study was approved by the Ethics Committee Università degli Studi della Campania “Luigi Vanvitelli”—A.O.U. “Luigi Vanvitelli”, A.O.R.N. “Ospedali dei Colli” ID 181060320. All methods were performed following the relevant guidelines and regulations, and informed consent was obtained from all subjects and/or their legal guardian(s).The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.The primary endpoint was the time to the clinician's evaluation of true relevant arrhythmias automatically detected by ILR. The following events, irrespective of symptoms, were considered : MobitzTo control bias in comparisons, the two study groups were balanced with respect to 30 potential confounding variables , using the propensity score as described below.t test or Mann–Whitney U test depending on the result of the normality test. Categorical variables were compared with Pearson's Chi-squared test or Fisher exact test, when appropriate.Continuous variables were reported as mean ± standard deviation if normally distributed, or as median (interquartile range) if non-normally distributed. Normality was tested with the Shapiro–Wilk method. Binary or categorical variables were reported as count . Comparisons of continuous variables were performed with the Student’s Distributions of times to event evaluations were plotted with the Kaplan–Meier method and the hazard ratios (HRs) in the RM-ON vs. RM-OFF groups were reported as a measure of ratios of the rate of event evaluations between study groups.Survival analysis was performed for primary and secondary endpoint events, computing event-free rates by study groups (RM-ON vs. RM-OFF) using uni- and multivariate Cox proportional hazard models. To reduce uncontrolled biases due to the nonrandomized study design, we used the PS to match the study groups with the inverse probability of the treatment weighting method . BalanciAnalyses were performed with R software version 4.1.0 . Propensity scores were calculated with the WeightIt package (version 0.12.0).p = 0.007).A total of 241 patients  were included in the study; 133 (55.2%) were in the RM-ON group and 108 (44.8%) were in the RM-OFF group. The clinical characteristics of the study population are summarized in However, after PS matching, absolute standardized mean difference of all the 30 variables was reduced to 10% or less , except During a median follow-up of 22 (12–34) months, arrhythmias were detected in 38 patients (28.6%) in the RM-ON group and in 22 patients (20.4%) in the RM-OFF group. Overall, 46 events (76.7%) were sinus bradycardias/arrests or atrioventricular blocks according to types 1 and 2 of the ISSUE classification. In the RM-ON group, post-diagnosis medical interventions were pacemaker implantations in 25 patients (18.8%), supraventricular tachycardia ablation in 1 patient (0.8%), and therapy change/adjustment in 3 patients (2.2%). In the RM-OFF group, there were 14 pacemaker implantations (13.0%), 2 supraventricular tachycardia ablations (1.8%), and 6 therapy change/adjustments (5.5%). Conservative measures, including hydration, salt consumption, reducing the hypotensive agents, and recognition of prodromal symptoms, were always recommended in patients with suspected typical reflex syncope. Pacemaker implantation was considered after screening according to age (>40 years) and duration of the ILR-recorded asystole recorded , as suggested by the guidelines.ILR-detected events eliciting diagnosis were asymptomatic in 24 patients (18.0%) in the RM-ON group and in 8 (7.4%) in the RM-OFF group, eventually leading to pacemaker implantation in 12 (9.0%) in the RM-ON group and in 2 (1.9%) in the RM-OFF group .p = 0.005), reflecting a 2.5-fold higher rate of event evaluations in the RM-ON group as compared to the RM-OFF group  in the RM-ON vs. RM-OFF group , reflecting a significantly shorter time in the RM-ON group [median 54 days (14–83)] as compared to the RM-OFF [median 89 days (32–306)]  months were available in the 29 patients of the RM-ON group who reached the study endpoint and received treatment. Of this subgroup, two patients of this subgroup (7%) experienced syncopal recurrence: one had received a pacemaker for symptomatic asystole; one had received a pacemaker for asymptomatic atrioventricular block.In our PS-matched comparison of remotely monitored patients who received an ILR for unexplained syncope with a historical cohort of non-remotely monitored patients, RM enhanced the diagnostic power of ILR with a 2.5-fold higher probability of clinician's evaluation of arrhythmic episodes and a median 46 days earlier diagnosis vs. a biannual schedule of in-office visits (50% relative reduction). Our estimations were obtained by balancing the two groups with the PS-match method based on the inverse probability of treatment weighting . The metThe RM effect was almost double if events were asymptomatic  with about 85% reduction in median time to diagnosis (35 vs. 240 days). The chance of a diagnosis of events leading to the indication of pacemaker implantation was also 2.8-fold higher with RM-ON vs. RM-OFF.Syncope is a common medical problem with a frequency between 15% and 39% in the general population and an annual number of episodes of 18.1 to 39.7 per 1,000 patients . Reflex In one observational study using weekly remote transmissions from ILR in patients with unexplained syncope , RM alloFast diagnosis is important for several reasons, including early therapy initiation, safety, and quality of life improvement. Moreover, it also helps reduce the duration of long-term monitoring and the need for device replacements while waiting for the diagnosis. Diagnostic rates have been reported to be 43% still after 2 years, and 26% of all achieved diagnoses occurred after 18 months from implantation . A 2.5-fA 50% reduction in time to event evaluation is not unexpected with RM. With accurate alert programming and daily remote transmissions, RM enables immediate within 24 h) notification of relevant episodes. Therefore, assuming a uniform distribution of events over time, the expected average time to diagnosis using RM should be about 50% of the between-visit interval of an in-office visit program. The device and alert programming we used should cover a wide range of arrhythmic events potentially underlying syncope  and may be easily replicated in any available device and RM system. However, the improvement of the diagnostic power of ILR conferred by RM may significantly depend on compliance and reliability of remote transmissions. In our study, we used an RM technology known to ensure daily transmissions with about 90% rates of successful transmissions , 14, and h notifiOur study has several limitations, including the lack of randomization, the relatively small sample, and the use of a historical cohort as control group. In the RM-ON group, 25 of 32 patients with sinus arrest, >10 s bradycardia, or atrioventricular block underwent pacemaker implantation according to current guidelines. Many of them did not report symptoms in relation to the RM-detected arrhythmias, so the direct relation of clinical syncope with these arrhythmias could not be demonstrated. The clinical relevance of subclinical ILR-detected arrhythmias has been recently questioned in patients with cryptogenic stroke by the results of a subanalysis of the Loop Recorder Detection of Atrial Fibrillation to Prevent Stroke (LOOP) study, showing that the risk of syncope and sudden death was similar in patients with and without ILR, despite the significantly higher incidence of bradyarrhythmias detected in the ILR group . HoweverFinally, the estimated benefit of RM we observed may be in part ascribed also to a general improvement of medical care over time that we could not factor in, as our comparison was between two cohorts observed in different years. Our results should be confirmed by randomized studies.In our PS-matched controlled study, RM of ILR in patients with unexplained syncope was associated with a 2.5-fold higher chance of evaluations of arrhythmias underlying syncope (>4-fold higher in asymptomatic episodes and 2.8 in arrhythmias justifying pacemaker implantation) as compared to conventional follow-up with biannual in-office visits.